The present invention relates to a process and apparatus for continuously propagating microorganisms in a culture medium. More particularly, the present invention relates to a process for continuously propagating microorganisms whereby a crude or precursor culture medium for the microorganism is continuously treated, as an intimate part of the propagation process and apparatus, to provide a suitable purified culture medium for the microorganism.
Numerous biochemical processes are known wherein a microorganism is propagated in a suitable culture medium therefor, either for the purpose of growing large quantities of the microorganism for some particular ultimate purpose or use, or for recovering products produced by the growing microorganism.
Economics and technical considerations generally favor the utilization of a continuous process for these purposes, but, with few exceptions, commercial microorganism propagation processes are conducted as batch or semi-batch processes. Continuous processing has proven to be difficult and, indeed, undesirable for many microorganism propagation systems owing to the inability, among others, to achieve the degree of control required in such processes. This is particularly true with respect to control over the concentration of gaseous materials necessary, or desirably present, in the propagation process, such as oxygen in aerobic propagation systems and oxygen and/or other gases employed in anaerobic systems either to promote or suppress by-product formation, provide suitable gas tensions, or other like functions. In particular, the nature of many microorganism propagation systems is such that the conditions at which they are conducted generally are not particularly conducive to significant solubility of gases in the nutrient or culture medium. As a consequence, gas (e.g., oxygen) utilization in the propagation system is generally quite poor, and resort to use of substantial (e.g., 100 to 1000 fold) excesses above theoretically required gas quantities is not atypical. The need to employ such large quantities of gaseous materials adds considerably to the difficulty of performing the process in a continuous mode and, of course, adds considerably to the expense of the overall process.
Another significant limitation on the possible use of continuous processing in microorganism propagation systems is the substantially universal requirement that the culture medium be purified to remove therefrom microorganisms or other materials which might contaminate the desired microorganisms or the products sought to be recovered therefrom. The degree of purification needed is generally quite high and may involve a number of heat treatments, filtrations or other means for removing undesired materials from the culture medium. In such circumstances, it is quite difficult to develop a process which, from medium purification through microorganism propagation, is truly continuous.
An excellent example of an industry where, despite obvious economic advantages, adoption of a continuous process has been problematic, is the commercial production of yeast.
Commercial yeast production typically is a batch process which entails propagation in a plurality of stages. Generally, yeast are inoculated into a presterilized nutrient medium usually contained in a shaker flask. In the flask, growth of the yeast is encouraged by various means such as controlling the temperature and shaking the flask to effect aeration. The yeast are removed from this flask and inoculated into another flask containing a larger volume of nutrient medium for continued growth. These initial stages may conveniently be referred to as flask or culture development stages.
From the culture development stages, the yeast may be inoculated into a vessel having an air source and means of agitation. These steps may be repeated once or twice using greater amounts of nutrient medium and larger vessels. Because the amount of air used in these stages is generally restricted, these stages are commonly referred to as slightly aerobic stages. Yeast from these stages are then transferred into larger fermentors where vigorous growth conditions are maintained, including the use of large volumes of air. These stages may be referred to as highly aerobic, or commercial, stages since the yeast from these stages are harvested and processed for bakery or home use, typically in compressed or active dry form.
For propagation in the highly aerobic or commercial stages, it is necessary to prepare large quantities of a yeast culture medium which is substantially free of microorganisms. This has been accomplished in the past by sterilizing the medium, such as final molasses, by heat treatment. To reduce the count of contaminating microorganisms to a level effective to produce yeast suitable for food use, large amounts of energy, as well as means for generating and transferring heat to the process, were required. Typically, the heat was generated in oil or gas-fired boilers and transferred to the process as steam which could be injected live or transferred by means of heat exchangers. Subsequent to heating, the molasses would then require cooling prior to use. Thus, this sterilization step entailed sizable capital and operational costs.
In commonly-assigned U.S. Pat. No. 4,379,845 of Apr. 12, 1983, there is disclosed an improved method which eliminates the need for the above-noted thermal sterilization and offers other improvements as well. That method, in its broad aspects, comprises purifying molasses by passing the molasses through an ultrafiltration device (which can be a spirally-wound membrane) effective to reject solids having molecular weights greater than about 30,000 daltons to produce a first permeate, and then passing the first permeate through at least one additional filtration device (which can be a tubular membrane) having an average pore diameter of from about 0.2 to about 1.2 microns to produce a yeast culture medium. The filtration devices are effective in combination to reduce the microorganism count to a level effective to produce yeast suitable for food use. The yeast culture is then inoculated with yeast in a suitable reaction vessel, and the yeast and the yeast culture medium are then subjected to conditions effective to propagate the yeast.
The method of U.S. Pat. No. 4,379,845 provides a significant advance over the prior art with respect to providing a purified culture medium for yeast propagation. The teachings of U.S. Pat. No. 4,379,845 more readily lend themselves to continuous production of yeast than processes theretofore known in the art, but there exists a need for providing a continuous bioreactor and process which would take greatest advantage of this improved method and solve other problems relating to the degree of control over propagation conditions which can be achieved in a continuous process.
Among the so-called continuous reactors presently known are those of Stich in U.S. Pat. Nos. 2,244,902 and 2,657,174, Ehnstrom in U.S. Pat. No. 3,940,492, and Fukuda et al in U.S. Pat. No. 4,284,724.
In U.S. Pat. No. 2,244,902, Stich discloses a process employing a number of interconnected reactors wherein each has means for establishing a vertically-circulating flow of yeast mash and means for introducing air into the downwardly moving portion of the mash. The yeast is circulated within each reactor for a number of cycles and is then transferred to another chamber. The method is said to improve the efficiency of introduction of air as compared to the known reactors wherein cells toward the upper part of the chamber receive relatively low levels of oxygen.
In U.S. Pat. No. 2,657,174, Stich discloses another method for continuous yeast manufacture. According to this method, a yeast mash is withdrawn from a plurality of locations near the bottom of a fermentation chamber, cooled, enriched with nutrients and reintroduced into the chamber at different locations. Within the chamber, the mash flows downwardly, countercurrent to the flow of air into the chambers. Again, the improvement is said to relate to improved oxygen distribution within the reactor. As with the earlier Stich patent, extremely large reactor volumes and separate sources of purified nutrient are required.
Ehnstrom, in U.S. Pat. No. 3,940,492, discloses a process wherein wort is continuously supplied to a circuit including an elongated closed channel through which microorganisms are fed. After fermentation has taken place in the circuit, the mixture of wort and microorganisms is centrifuged to separate it into fermented wort, a living cell mass and impurities. These three components are separately discharged from the centrifuge. The fermented wort and living cell mass are discharged continuously. The living cell mass includes an excess of living cells formed in the circuit. This excess is discharged from the circuit. As with the procedures of Stich, a separate source of sterilized nutrient is required to supply this complex apparatus.
According to the disclosure of Fukuda et al in U.S. Pat. No. 4,284,724, a broth of yeast cells is continously or intermittently removed from a fermentor. Yeast cells then are separated from the filtrate using a cell separator, or further washed with water. The yeast cells so obtained then are recycled to the fermentor, whereby yeasts are cultivated at a high cell concentration of from 6% to about 20% based on dry weight. It is disclosed that by removing the filtrate from the cultivation system, there is no accumulation of metabolities and salts prohibiting the cultivation of yeasts, and the growth of miscellaneous microorganisms which interfere with yeast cell growth is suppressed. As with the other systems, separate means are required to provide sterile nutrient.
There exists a definite need for apparatus and processes which could be employed in a continous operating mode for the propagation of microorganisms in a culture medium, which affords control of the propagation process to the high degree required and which provides for continous purification of culture medium as an intimate part of the process.